Phosphatide separation

ABSTRACT

N-acylphosphatides, for instance N-acetylcephalin, are separated from phosphatides without an acylatabe amino group, for instance lecithin, by making a mixture containing them acid to the equivalent of pH less than 3.5 under aqueous conditions and solvent-fractionating with acetone or methyl acetate.

This invention relates to a process for the separation of phosphatidesfrom mixtures containing them.

Phosphatide mixtures are obtained as by-products in the production ofvegetable oils and are used in the margarine industry for theiremulsifying properties in water-in-oil emulsions. Such phosphatidemixtures consist of various phosphatides, and especially ethanolamine,serine, inositol and choline phosphatides, together with neutraltriglyceride oil, free fatty acids, water and other accompanyingsubstances, including small quantities of steroids. The amounts oftriglyceride oil present in commericial phosphatides are often withinthe region of 30 to 40% by weight of the total material, and theproducts are viscous liquids. In commerce mixtures of these phosphatidesare often referred to as lecithin, but this name is also used morespecifically for the choline phosphatide itself, and it is in thislatter sense that lecithin is used in this specification.

Ethanolamine and serine phosphatides have the structure. ##EQU1## whereR and R' are long chain fatty acid radicals and R" is hydrogen andhydroxycarbonyl respectively: these phosphatides are together knownunder the name cephalin. Inositol phosphatides have the structure##EQU2## where R and R' are long chain fatty acid radicals and M is ametal atom, for instance sodium, potassium, calcium or magnesium, andthe inositol group may be substituted by glycoside and other radicals. Avery active emulsifier component of the phosphatide mixtures is thecholine phosphatide, lecithin, which has the structure ##EQU3## where Rand R' are long chain fatty acid radicals. All four phosphatides have anacid hydroxy group, shown above in salt form, but of the threenitrogenous phosphatides only lecithin has no free amino group.

Where cephalin and lecithin are present together in a phosphatidemixture, as in the by-products mentioned above, the cephalin has adeleterious effect on the emulsifying properties of the lecithin by somekind of antagonising action, for instance when used in margarine: otherundesirable characteristics have also been attributed to cephalin; thus,parenterally administered fat emulsions prepared using phosphatidescontaining cephalin have a hypertensive action for which the cephalinpresent is responsible, and when phosphatide mixtures are hydrogenatedcephalin present is commonly believed to have a deactivating effect onthe catalyst. On the other hand no disadvantage attends the presence ofinositol phosphatides.

Accordingly, methods have been sought for avoiding the deleteriouseffect of the cephalin. In one such process, described in British Pat.1,174,399, vegetable phosphatide mixtures are modified by reacting thefree amino group of the cepahlin present with an acylating agent. Thisforms acylcephalin which does not antagonise the emulsifying propertiesof lecithin, so that the phosphatide mixture, in which the acylcephalinremains present, has an improved emulsifying action.

Attempts have been made to separate cephalin from lecithin inphosphatide mixtures in order to obtain a phosphatide with a higherlecithin concentration having improved properties but this has proved adifficult problem. Solvent fractionation results in a partialseparation, providing lecithin-enriched and cephalin-enriched fractions,and British Pat. 1,113,241 describes an enrichment process of this kindusing an aqueous alcohol extraction by which the proportion of lecithinto cephalin is increased by from 1:1 to from 5:1 in the extract, butsubstantial proportions of lecithin remain in the residue.

British Pat. 1,217,846 describes one solution of the problem ofseparating cephalin from lecithin, where phosphatide mixtures containingthem are acylated to convert the cephalin present into acylcephalin andthe lecithin and acylcephalin are separated by solvent fractionationwith acetone or methyl acetate in the presence of sufficient of anacetone-soluble or methyl acetate-soluble nitrogen base to provide basicconditions equivalent to a pH in water of at least 8.5. While thisrepresents a marked advance it has the disadvantage that traces ofnitrogen base remain in the separated phosphatides after removal ofsolvent, and such residues of nitrogen bases, which are generallyundesirable where the product is to be used in foodstuffs, cannot beremoved completely and with certainty without recourse to additionalsteps which raise the cost of the process.

The present invention is concerned with another method of separatingN-acylphosphatides such as N-acylcephalins from non-N-acylatablephosphatides in which nitrogen bases are not employed, and no residuesof them therefore remain.

It has now surprisingly been discovered that an acylcephalin can bereadily separated from lecithin or inositol phosphatide in mixturescontaining them by solvent fractionation with acetone or methyl acetatein the presence of sufficient of an acid to provide an acidityequivalent to a pH under aqueous conditions of less than 3.5. By acidity"equivalent to a pH under aqueous conditions" of a given value it ismeant that the acidity of the composition is such at a 1% by weightdispersion of the composition in water has that pH value. These areconditions very different from those of British Pat. 1,217,846, and themechanism of action of the process is not understood. The acylcephalinis extracted by the solvent, leaving the lecithin or inositolphosphatide behind. Unacylated cephalin cannot be separated from theother phosphatides under these conditions. The separation is alsoeffective in the presence of large amounts of triglyceride oil such asare present in commerical phosphatides; triglycerides are removed withthe cepahlin.

Accordingly, the present invention provides a process for separating anN-acylphosphatide and a phosphatide without an acylatable amino groupfrom a mixture comprising them, which comprises adding to the mixture asufficient amount of an acid to provide the equivalent of pH less than3.5 under aqueous conditions, extracting the mixture with acetone ormethyl acetate, and separating the phases containing theN-acylphosphatide and the phosphatide without an acylatable amino group.

In practice a phosphatide mixture that comprises N-acylatable andnon-N-acylatable phosphatides is contacted with an acylating agent toconvert the N-acylatable phosphatide to N-acylphosphatide, a sufficientamount of an acid to provide the equivalent of pH less than 3.5 underaqueous conditions is added, the mixture is extracted with acetone ormethyl acetate, and the phases containing the N-acylphosphatide and thenon-N-acylatable phosphatide are separated.

In practice the N-acylatable phosphatide is cephalin, and at least somelecithin is preferably present as non-acylatable phosphatide; forinstance the phosphatide mixture can comprise cephalin, lecithin andinositol phosphatide. As phosphatide starting materials for theseparation process there can be used commercial phosphatides, forinstance the hydrated vegetable phosphatides obtained by treating with asmall amount of water a phosphatide-containing crude oil extracted fromplants, for example crude soyabean oil, so that the phosphatides becomehydrated and are precipitated as a gum and are then separated. Egg yolkphosphatides can also be used. Preferably the phosphatide mixturecontains from 0.1 to 20 parts of lecithin to each part by weight ofcephalin. Such phosphatides and modifications of them that can be usedare described in British Pat. 1,118,373. Lecithin-enriched orcephalin-enriched phosphatides obtained by solvent fractionation withaqueous alcohols as described in British Pat. 1,113,241 can be used.Vegetable phosphatide compositions containing up to 80%, for instancefrom 20 to 60%, by weight of triglyceride oil can be used. The acylatedphosphatide mixtures obtained as described in British Pat. 1,174,399 canalso be employed. Partially hydrogenated phosphatide mixtures can beused where the original fatty acyl groups included those of unsaturatedfatty acids and some of these have been saturated by hydrogenation. Thephosphatide starting materials can contain appreciable quantities ofwater even before the acylation step, for it has been found that thepresence of water does not seriously interfere with N-acylation andhelps to prevent O-acylation by excess acylating reagent which wouldgive rise to phosphatides in which the hydroxy groups of inositolphosphatides present, as well as the hydroxy group attached to thephosphorus atoms, are acylated: acylation at the phosphorus atom causesno problem as the acyl group can be removed by after-treatment withwater, but the acylation of inositol groups can represent an unnecessaryloss of acylating agent, for inositol phosphatides can be present inlarge proportions: also, once acylated, the inositol hydroxy groupsconcerned cannot be re-formed because the conditions necessary for thiswould remove the acyl groups esterifying the glyceryl radical and thusdestroy the phosphatides.

As acylating agent there is preferably used a carboxylic acid anhydride,especially a fatty acid anhydride, for example acetic, propionic,capric, oleic or stearic anhydrides, or mixed anhydrides such as thoseobtained from soyabean fatty acids by trans-anhydridisation with aceticanhydride: maleic, succinic, or phthalic anhydride can also be used.Other acylating agents, for instance acyl chlorides such as acetylchloride, and ketene, can be used, but as they react more readily withwater and with hydroxy groups of substances present in the startingmaterials, they are less efficient than anhydrides. The amount ofacylating agent required is that necessary to acylate all the free aminogroups present, with allowance for loss of reagent where water presentcompetes by hydrolysis. As phosphatides rarely contain more than 30% byweight of cephalin, 4% of acetic anhydride by weight of phosphatide isusually satisfactory. Where the phosphatide starting material is highlyviscous, reaction can be carried out in a suitable inert solvent medium,for instance in hexane, benzene, or chloroform, or even in acetone ormethyl acetate where substantial amounts of triglyceride are present,but no solvent is generally necessary where the phosphatide mixturecontains sufficient triglyceride oils to make it fluid. The mixtureshould be stirred during reaction, and this is especially desirablewhere no solvent is employed. The acylation can be conducted in an inertatmosphere, for instance under nitrogen.

As free acid derived from the acylating agent is progressively formedduring the reaction where the acylating agent is an anhydride, and thisfree acid tends to inhibit completion of acylation by suppression of thereactive unprotonated amine form of the remaining N-acylatablephosphatide in favour of the unreactive protonated form it is preferableto promote completion of the acylation by removal from the reactionmixture of the free fatty acid formed. This can be done by physicalmeans, for instance, distillation: thus the acid can be distilled offeither as an azeotrope, for instance that with carbon tetrachloridewhich distills at 76.5° C. and contains 3% acetic acid, or simply byevaporation under reduced pressure. If any anhydride needed foracylation is removed at the same time, it can be replaced. Particularlysuitable where acetic anhydride is used as acylating agent is reactionof a suitable excess, for instance 33%, at 80° C. under a reducedpressure of 25 mm. of mercury maintained with a solid potassiumhydroxide trap. In this way a reaction product forms in which more than95% of the cephalin originally present has been converted toacylcephalin. A typical product of acylation, after removal of aceticacid by such evaporation, gives a 1% aqueous dispersion of pH 6.3.

The progress of the acylation reaction can be followed by thin layerchromatography on silica gel plates with as mobile phase a mixture ofchloroform, methanol and water in proportions of 65:25:4 by volume, andtreatment of the developed chromatogram with ninhydrin reagent. Reactionis complete when the pink spot of R_(f) about 0.6 due to free primaryamino compound is no longer visible. Preliminary tests can be carriedout using this technique to determine the amount of acylating agent andother conditions most appropriate for use in acylating any particularphosphatide mixture, for instance a mixture of unknown N-acylatablephosphatide content.

After acylation the excess acylating agent can be neutralised, or wherethe acylating agent is volatile, it also can be evaporated off, forinstance under reduced pressure. Any solvent which would interfere withthe subsequent fractionation can also be removed by evaporation.

In the solvent fractionation step, which will normally consist ofextracting with the solvent and separating the phases containing theN-acylphosphatide without an acylatable amino group, acetone or methylacetate (or a mixture of them) is used at the equivalent (under aqueousconditions) of pH less than 3.5, and preferably pH 2 to 3, provided bythe presence of sufficient of a suitable acid. The acidity can beprovided by the addition of a suitable strong acid; the amount of thisnecessary can be determined by testing the quantity of acid needed togive the required pH when a sample of the acylated phosphatidecomposition is dispersed in water at 1% by weight concentration, suchconcentration being exclusive of any solvent that is present.Particularly suitable acids are those of pK_(a) less than 3, andespecially less than 2.5: they are preferably inorganic acids,especially hydrochloric, sulphuric, phosphorous, and orthophosphoricacids, whose pK_(a) 's are --3, 1.9, 2.0 and 2.1, respectively. Strongorganic acids such as tartaric acid, pK_(a) 3.0, and trichloroaceticacid, pK_(a) 0.7, can also be used. Where the product is to be used infood, the acid will of course be an edible one. The acid can beintroduced into the phosphatide mixture before the extraction, or intothe solvent used for the extraction.

The fractionation step can be carried out using the standard procedureof acetone extraction for determination of acetone-insoluble matter inphosphatides described in the American Oil Chemists' Society OfficialMethod Ja 4-46. The acetone extraction removes acylcephalin and anytriglyceride oil present, leaving the lecithin-rich fraction as residue.It is preferable to dilute the acylated phosphatide mixture with a smallamount of acetone to give a homogeneous mixture and to add this slowlyto the bulk of acetone in order to achieve the most rapid extraction.The material to be fractionated can be extracted several times withacetone. Where there is more than one extraction, it is the initialcondition of acidity that governs the efficiency of the extraction.Methyl acetate can be used similarly. Extraction is preferably carriedout at room temperature.

Although a total separation of acylcephalin and lecithin may not beachieved in any particular case, a much more complete fractionation iseffected than when attempts are made to separate the components withacetone under neutral conditions. The residues from extraction containvery little acylcephalin and are obtained as straw-coloured solids whichon grinding form free-flowing powders that are readily dispersible inwater or triglyceride oils. They are particularly valuable asemulsifiers and dispersing agent. The acylcephalin produced can berecovered from the acetone extracts after removal of acetone by suitablemethods, for instance evaporation: the acylcephalins can also be used asemulsifier. Where the amount of residual acid in a product from theextraction is undesirably high it can be neutralised. An acylcephalincan be reconverted to cephalin where the acyl group is of such a naturethat it can be removed without destruction of the cephalin: thus ifacylation is effected with phthalic anhydride, the resultingo-carboxybenzoylcephalin can be cyclised by heating or the action ofacetic anhydride to give phthaloylcephalin and cephalin liberated fromthis by heating with hydrazine.

Those products of the process of the invention that contain bothlecithin and inositol phosphatides can be further fractionated withalcohol to provide alcohol-soluble fractions enriched in lecithin andalcohol-insoluble fractions enriched in inositol phosphatides.

The invention is illustrated by the following examples, in which alltemperatures are in ° C., pressures are in mm. of mercury, the acetoneused contained about 1% of water by weight, and acetone-insolubles weredetermined by the A.O.C.S. method referred to above. TLC is thin layerchromatography.

EXAMPLE 1

A commercial unbleached Canadian soya phosphatide of average gradehaving the following approximate composition was used as startingmaterial.

    ______________________________________                                                             Parts by weight                                          Triglycerides        40                                                                            =                                                        Total acetone-insolubles                                                                           60                                                                            --                                                       Lecithin             15                                                       Cephalin             12                                                       Inosital phosphatides                                                                              23                                                       Sugars, steroids and salts                                                                         10                                                                            =                                                        Water                 1                                                       Total phosphorus content                                                                           2.2                                                      ______________________________________                                    

This phosphatide (100 g.) and acetic anhydrate (4 g.) were stirredtogether vigorously at 80° (bath temperature) for 2 hours under 25 mm.pressure maintained through a trap of potassium hydroxide pellets. Thecooled acylated mixture was stirred with acetone (50 ml.) containingorthophosphoric acid (88%, 2.5 g.) and the mixture obtained, which hadan acidity corresponding to pH 2.95 in an aqueous solution containing 1%by weight of the mixture exclusive of the acetone, was allowed to dripslowly during 1 hour into acetone (1 litre) with vigorous stirring,which was maintained for a further half hour. The acetone, containingN-acetylcephalin and triglycerides, was decanted off and further acetone(1 litre) added and the mixture stirred for half an hour, and thisdecantation and solvent extraction step repeated. The residue wasfiltered off, washed with acetone (150 ml.), and residual solventremoved under reduced pressure at 25 mm. and 40° for one hour.

The product (39.2 g.) was a pale cream solid which contained 28% byweight of lecithin and 50% of inositol phosphatides, and showed (TLC)only traces of N-acetylcephalin and cephalin.

EXAMPLE 2

The process of Example 1 as repeated with substitution of theorthophosphoric acid by concentrated hydrochloric acid (35%, 1.0 g.)giving an initial extraction mixture with an acidity equivalent to pH2.9 under aqueous conditions. The final residue (40 g.) contained onlytraces of N-acetylcephalin and cephalin (TLC).

EXAMPLE 3

Example 2 was repeated, but using 3.0 g. 35% hydrochloric acid, with amixture having an acidity corresponding to pH 2.3. The product (36 g.)contained no N-acetylcephalin or cephalin.

EXAMPLE 4

Example 1 was repeated using, instead of the commercial Canadian soyaphosphatide, a commercial alcohol-insoluble fraction prepared from soyaphosphatide by extracting 1 part by weight of phosphatide with 3.5 partsof aqueous 90% ethanol at room temperature for a few minutes, decantingoff the ethanol and taking the residue, which had the followingapproximate composition.

    ______________________________________                                                             Parts by weight                                          Triglycerides        41                                                                            =                                                        Total acetone-insolubles                                                                           59                                                                            --                                                       Lecithin             12                                                       Cephalin             12                                                       Inositol phosphatides                                                                              29                                                       Sugar, steroids and salts                                                                           6                                                       Water                 1                                                       ______________________________________                                    

The same amounts of materials were used as in Example 1, except that 6g. acetic anhydride and 3.75 g. orthophosphoric acid (88%) wereemployed, providing an acidity equivalent to pH 2.8 under aqueousconditions.

From the phosphatide starting material (100 g.) there was obtained apale cream solid product (35.4 g.) containing lecithin and inositolphosphatides and only traces of cephalin and N-acetylcephalin.

EXAMPLE 5

A commercial rapeseed phosphatide having the following approximatecomposition was used as starting material.

    ______________________________________                                                             Parts by weight                                          Triglycerides        30                                                                            =                                                        Total acetone-insolubles                                                                           70                                                                            --                                                       Lecithin             14                                                       Cephalin              7                                                       Inositol phosphatides                                                                              32                                                       Sugars, steroids and salts                                                                         17                                                                            =                                                        Water                <1                                                       ______________________________________                                    

The phosphatide mixture (100 g.) was stirred with acetic anhydride (3g.) and heated at 80° for 2 hours under 25 mm. pressure maintainedthrough a trap of potassium hydroxide pellets. Into a portion of thecooled product (20 g.) was mixed concentrated hydrochloric acid (35 %,1.0 g.) and the mixture extracted with acetone as in Example 1. Theinitial extraction mixture had an acidity equivalent to pH 2.7 underaqueous conditions.

The residual composition (8.6 g.) contained lecithin, inositolphosphatides, and only traces of cephalin and N-acetylcephalin (TLC).

EXAMPLE 6

A commercial unbleached soya phosphatide having the followingapproximate composition was used.

    ______________________________________                                                             Parts by weight                                          Triglycerides        35                                                                            =                                                        Total acetone-insolubles                                                                           65                                                                            --                                                       Lecithin             15                                                       Cephalin             15                                                       Inositol phosphatides                                                                              23                                                       Sugars, steroids and salt                                                                          12                                                                            =                                                        Water                 1                                                       ______________________________________                                    

This phosphatide (250 g.) and acetic anhydride (13.5 g.) were mixedintimately and heated at 80° for 2 hours under 25 mm. pressuremaintained through a trap of potassium hydroxide pellets. The reactionmixture was allowed to cool to room temperature.

A portion of the acetylated mixture (25 g.) was stirred with acetone (10ml.) containing trichloroacetic acid (1.25 g.) and the mixture (acidityequivalent to pH less than 3.5) was allowed to drip slowly into acetone(250 ml.) while stirring vigorously. After the addition the stirring wascontinued for half an hour, the mixture then allowed to settle for 10minutes and the acetone decanted off. More acetone (250 ml.) was addedand the mixture stirred vigorously for half an hour. The mixture wasallowed to settle for 10 minutes and the acetone again decanted off.Fresh acetone (250 ml.) was added and the mixture again stirred. It wasnow filtered and the residue washed with acetone (50 ml.) and solventremoved at 40° under 25 mm. pressure for one hour. The product (10.5 g.)contained lecithin and inositol phosphatides, but no cephalin and only avery small amount of N-acetylcephalin (TLC).

EXAMPLE 7

Another portion of the acetylated phosphatide reaction mixture ofExample 6 (25 g.) was stirred with acetone (12 ml.) and the resultingsolution allowed to drip slowly into acetone (350 ml.) containing 50%aqueous sulphuric acid (1.5 g.). The resulting mixture had an acidityequivalent to pH 2.45 under aqueous conditions at a 1% concentrationexclusive of the solvent. The extraction procedure of Example 6 wasfollowed except that the further quantities of acetone were 250, 125 and50 ml. respectively. The product (10 g.) contained only traces ofN-acetylcephalin and cephalin.

What is claimed is:
 1. A process for separating an N-acylcephalin and aphosphatide without an acylatable amino group from a mixture comprisingthem which comprises adding to the mixture a sufficient amount of anacid to provide the equivalent of pH less than 3.5 under aqueousconditions, extracting the mixture with acetone or methyl acetate, andseparating the phases containing the N-acylcephalin and the phosphatidewithout an acylatable amino group.
 2. A process for the treatment ofphosphatides, which comprises contacting a phosphatide mixture thatcomprises cephalin .[.as.]. .Iadd.and .Iaddend.a non-N-acylatablephosphatide with an acylating agent to convert the cephalin toN-acylcephalin, adding to .Iadd.the .Iaddend.reaction mixture asufficient amount of an acid to provide the equivalent of pH less than3.5 under aqueous conditions, extracting the mixture with acetone ormethyl acetate, and separating the phases containing the.[.N-acylphosphatide.]. .Iadd.N-acylcephalin .Iaddend.and the.[.N-acylcephalin.]. .Iadd.non-N-acylatable .Iaddend.phosphatide.
 3. Aprocess according to claim 2, wherein the phosphatide mixture comprisescephalin and lecithin.
 4. A process according to claim 3, wherein thephosphatide mixture contains from 0.1 to 20 parts of lecithin to eachpart by weight of cephalin.
 5. A process according to claim 3, whereinthe acylating agent is a fatty acid anhydride.
 6. A process according toclaim 5, wherein the acid anhydride is acetic anhydride.
 7. A processaccording to claim 5, wherein the completion of acylation is promoted byremoval from the reaction mixture of the free fatty acid formed.
 8. Aprocess according to claim 7, wherein free fatty acid formed is removedby distillation.
 9. A process according to claim 8, wherein the acid isremoved by evaporation under reduced pressure.
 10. A process accordingto claim 1, wherein the extraction solvent is acetone.
 11. A processaccording to claim 1, wherein the reaction mixture containingN-acylcephalin is made acid to the equivalent of pH from 2 to 3 underaqueous conditions.
 12. A process according to claim 11, wherein theacid pH is provided by an acid of pK_(a) less than 2.5.
 13. A processaccording to claim 1, wherein the acid pH is provided by hydrochloricacid.
 14. A process according to claim 1, wherein the acid pH isprovided by orthophosphoric acid.